Controlled environment culture system for light microscopy

ABSTRACT

A cell culture chamber for use in optical microscopic techniques and equipment is provided with an apertured holder carrying two coverglasses mounted strain free and separated by a stainless steel spacer with tubes permanently attached therethrough. A pressure plate holds the coverglasses in place by being compressed by a sealing ring and a removable snap ring.

Unite States Patent [191 Dvorak et al.

CONTROLLED ENVRONMENT CULTURE SYSTEM FOR LIGHT MICROSCOPY Inventors:James A. Dvorak; Woodrow F. Stotler, both of Bethesda, Md.

The United States of America as represented by the Secretary of theDepartment of Health, Education and Welfare Filed: Mar. 9, 1972 Appl.No.: 233,194

Assignee:

US. (:1 ..356/244, 350/92 Int. Cl. ..-....G01n 21/16 Field Of Search..356/244, 246;

[451 Apr. 10, 1973 [56] References Cited UNITED STATES PATENTS 2,940,3606 1960 Carter, Jr. ..350/95 2,942,520 6/1960 Rose 3,220,300 lI/1965 VonHuene ..350/92 Primary ExaminerWil1iam L. Sikes AttorneyAlvin Browdy etal.

[57] ABSTRACT A cell culture chamber for use in optical microscopictechniques and equipment is provided with an apertured holder carryingtwo coverglasses mounted strain free and separated by a stainless steelspacer with tubes permanently attached therethrough. A pressure plateholds the coverglasses in place by being compressed by a sealing ringand a removable snap ring.

8 Claims, 2 Drawing Figures CONTROLLED ENVIRONMENT CULTURE SYSTEM FORLIGHT MICROSCOPY FIELD OF INVENTION The present invention relates tocell culturing, and, more particularly, to a cell culture chamber whichhas optically flat, strain-free, parallel surfaces with a geometricthickness usable for all extant optical techniques and equipment andwhich is a completely closed system to safely observe, handle andcontain living cells or metazoan organisms including human pathogens.

BACKGROUND OF THE INVENTION Biologists have long been concerned withfollowing the growth of a single cell by direct microscopic observation.Accordingly, numerous culture chambers have been developed for use withthe light microscope to study living cells or metazoan organisms. Someof the early prior art systems used concave slides for hangingdropcultures. For many cell types this kind of culture is inadequate fornormal cell growth as they require periodic replenishment of both gasand liquid phases of the growth medium. Furthermore, a concavity slideintroduces adverse optical effects in the light microscope.

Thus, micro-culture chambers were developed through which aerated growthmedium could be constantly perfused. Certain characteristics areextremely desirable in such a microperfusion chamber and heretofore noprior art chamber has satisfactorily fulfilled all of theserequirements. To be entirely satisfactory, a culture chamber would haveto have the following characteristics:

1. Usable with all extant transmitted light microscopy techniquesincluding bright field and phase contrast microscopy (Zernike) with highnumerical aperture, short working distance objectives and condensers;double beam quantitative interference microscopy (lamin- Lebedeff orMach-Zehnder); differential interference contrast microscopy (Nomarski);and polarizing microscopy. This necessitates that the assembled chamberhave optically flat, strain-free, parallel surfaces with a fixedgeometric viewing thickness not exceeding 1.2 mm.

2. A completely closed system to safely observe, handle and containhuman pathogens or other hazardous materials.

3. Sterilizability of the assembled chamber both prior to use and as ameans of decontamination at the end of the experiment.

4. Durable construction with biologically inert, non toxic materials.

5. Design simplicity permitting rapid and easy cleaning and assembly ofthe chamber.

6. The ability, after removal of the chamber from the microscope, torapidly relocate cells or other objects of interest from a fixed pointof reference.

7. Long-term maintenance of optimum physiologic conditions for the cellsor organisms being studied.

8. Rapid exchange or replacement of the culture media to observe andstudy the effects of varying physiologic parameters as well as theinstantaneous fixation of the cells for subsequent procedures such aselectron microscopy, immunofluorescence, histochemistry orautoradiography.

The microperfusion chamber described by Poynton and Branton inExperimental Cell Research (1970) 109-1 14 attempts to solve many of theabove problems but the resultant chamber is not entirely satisfactory.The Poynton device as well as many other prior art devices rely uponcompressible spacers to maintain the space between the glass surfaces ofthe chamber. The two pieces of glass are placed on either side of thespacer and then set screws or similar devices are tightened around theperiphery of the chamber to produce a seal. However, it is impossible totighten screws or similar devices uniformly to produce a chamber whichis truly strain free and in which the two pieces of glass are preciselyparallel.

Another disadvantage of prior artdevices is that when completelyassembled, the chamber is sealed so that if autoclaved they wouldexplode. The prior art devices must be autoclaved, disassembled and thenassembled under sterile conditions which is difficult or, at best, theycan be autoclaved partially assembled and the assembly can be completedafter autoclaving, thus increasing the danger of contaminating thechamber.

SUMMARY OF THE INVENTION Accordingly, it is an object of the presentinvention to overcome the deficiencies of the prior art.

It is another object to provide for improved and more efficient cellculturing.

It is a further object to provide an improved cell culture chamber.

It is another object to provide a cell culture chamber which hasoptically flat, strain free, parallel surfaces with a fixed geometricthickness usable for all extant optical techniques and methods.

It is another object to provide a cell culture chamber which operates asa completely closed system to safety observe, handle and contain humanpathogens or other hazardous materials, and is sterilizable as anassembled unit both prior to use and as a means of decontamination atthe end of the experiment.

It is still another object to provide a cell culture chamber whichrelies upon durable construction with biologically inert non-toxicmaterials and utilizes design simplicity to permit rapid and easycleaning and assembly of the chamber.

It is yet another object to permit rapid relocation of cells or otherobjects of interest from a fixed point of reference after removal of thechamber from the microscope.

It is another object of the present invention to provide a cell culturechamber which allows long-term maintenance of optimum physiologicconditions for the cells or organisms being studied and permits rapidexchange or replacement of the culture media to observe and study theeffects of varying physiologic parameters as well as the instantaneousfixation of the cells for subsequent procedures such as electronmicroscopy, immunofluorescene, histochemistry or autoradiography.

BRIEF DESCRIPTION OF THE DRAWINGS These and other objects will becomemore apparent from the following disclosure of an illustrativeembodiment taken in conjunction with the drawing in which:

FIG. 1 is a perspective view, partially cut away, of a cell culturechamber in accordance with the present invention; and

FIG. 2 is a partial cross-sectional view through a portion of theassembled cell culture chamber of FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to the drawing, aholder 11 having a serial identification number engraved on both facesis provided, preferably made of stainless steel, and having a platformportion 23. A lower coverglass 12 rests on the platform 23 which ispreferably coated with a thin layer 15 of Teflon(polytetrafluoroethylene). It is desirable that a Teflon layer be placedat all steel-glass interfaces to prevent the sharp steel edge fromacting as a die and cutting the coverglass under the influence of highperfusion pressures.

A second coverglass 14 is placed parallel to coverglass 12 and a spacer13; preferably of stainless steel, is placed between these coverglasses12 and 14. Such coverglasses are preferably free of lead oxide andshould have minimal strain or other optical defects; this isparticularly important when polarized light is used.

For some optical uses of the cell culture chamber it is highly importantthat the finished spacer 13 between the coverglasses 12 and 14 have athickness of 635 pm with a tolerance of 25 pm. This separation will givea total outside dimensional measurement of thickness between the twoglass surfaces of 1.2 mm maximum (using No. l 1% thicknesscoverglasses). This is the same thickness as a properly corrected glassmicroscope slide and coverglass. It permits microscopic examination ofcultured cells under the same optical conditions as those obtained usinga microscope slide and coverglass.

Suitable perfusion ports, preferably consisting of 27 gauge stainlesssteel tubing 20 fitted with stainless steel Luer-taper hubs 24, passthrough and are permanently bonded to the body of the spacer 13 with asolder containing no lead or cadmium, or other suitable adhesive. Bothsurfaces of the spacer 13 which contact the coverglasses 12 and 14 arecoated with an 80 pm layer of Teflon.

A pressure plate 16, suitably of stainless steel and having a steppedbottom surface which contacts the upper coverglass 14, is providedperipherally above the upper coverglass 14. This step, which ispreferably coated with an 80 um layer of Teflon, applies uniformpressure on the coverglass-spacer assembly 12, 13, 14.

A silicone rubber O-ring 17, having its outer diameter flat to fit therecessed well of the holder 11, is fitted over the pressure plate 16.The O-ring 17 may be made of any compressible material which isnon-toxic and dimensionally stable from approximately 60 to +230 C. Itmay be molded, machined and/or ground to the desired shape.

The entire assembly is sealed through use of a stainless steel snap ring18 which fits into a groove 19 in the holder 11. The combination of snapring 18 and O-ring 17 which is external to the cell'chamber rather thanbetween the glass surfaces permits predetermined pressures to beattained every time the snap ring 18 is snapped into place. There are noclamps or screws to adjust in order to obtain the necessary seal.

In the manufacture of the device all stainless steel parts arepreferably machined dry without oil lubricants. This is done becausecutting oils are toxic and cannot be practically removed from the steelafter machining has been completed.

Suitable stabilizer clamps 22 may also be included and located onopposite sides of the cell chamber. The clamps 22 support the Luer-taperhubs 24 used for connecting the perfusion tubing to the device and suchclamps 22 prevent the needles 20 extending through the stainless steelspacer ring 13 from being sheared off. This would ruin the chamber andwould permit the escape of the material being cultured, resulting in thepossible contamination of the investigator and the laboratory with humanpathogens. The stabilizer clamps 22 also act as handles which facilitatehandling of the cell chamber.

In order to facilitate relocation of cells or other objects of interestfrom a fixed point of reference, the holder 11 may have machined ontoone edge an alignment radius 25 which fits an alignment pin on themechanical stage plate of the microscope.

One method of assembly and operation will now. be described.

I. Selection and Preparation of Coverglasses 1. Select No. l lcoverglasses. They should be 170 m thick i 5 m if dry objectives withoutcorrection collars are to be used). Flat, strain-free coverglasses areessential when procedures employing polarized light are anticipated.

2. Place coverglasses in a l-INO -l-I O (1:1) solution, heat to C. in afume hood, turn off heat and allow to stand overnight.

3. Rinse coverglasses with tap water.

4. Place coverglasses in EDTA (versene) solution (1 g. EDTA/literdistilled water) 5. Rinse three times in distilled water.

6. Rinse two times in glass-distilled water.

7. Place coverglasses in absolute ethanol (99.5 percent) for one-halfhour.

8. Remove coverglasses individually from alcohol with clean,plastic-tipper forceps, wipe dry gently with lens paper held betweenthumb and forefinger with clean gauge pads to prevent skin oils fromcontacting alcohol or coverglass.

9. Place the dried and polished coverglasses in a clean Petri dish onfilter paper. Six coverglasses can be placed in a standard 90 mm Petridish.

10. Wrap Petri dish in aluminum foil to keep out dust.

II. Cleaning, Assembly and Sterilization of Chamber 1. Soak all chamberparts in diethyl ether for onehalf hour. Repeat twice using fresh ether.This operation removes microscope immersion oil from chamber parts.

2. Wash all parts of chamber in Hemosol solution at 71 C. with a stiffbrush (do not use the brush to clean the Teflon seals).

3. Rinse with tap water.

4. Rinse with running distilled water.

5. Pass one or two ccs of distilled water through each perfusion tube inthe spacer.

6. Place washed and rinsed chamber parts in a beaker of distilled waterand boil on a hot plate for onehalf hour.

7. Remove boiled chamber parts from water, place in open glass Petridishes and a dry in an oven at 121 C. for one-half hour.

8. Assemble chamber using clean forceps to handle chamber parts andplastic tipped forceps to handle coverglasses and Teflon seals. Theorder of assembly is as follows: Holder, Teflon seal, coverglass, Teflonseal, spacer, Teflon seal, coverglass, Teflon seal, pressure plate,silicone O-ring, and snap-ring. Make sure all components are seatedproperly before installing the snap-ring. Tighten the hubs in the hubholders after the snap-ring is installed.

9. Place the assembled chamber in a clean 90 mm.

Petri dish.

10. Sterilize the assembled chamber for six hours at 140 C. in a dryingoven. Wrap Petri dish in aluminum foil to keep out dust. Chamber is nowready to be used. Ill. Inoculation of Chamber with Cells NOTE: Steriletechnique should be used throughout this procedure.

1. Attach i.v. extension tubing (e.g., Bardic tubing) to outlet bottleleaving the male end covered. NOTE: Check for defects in the ends ofi.v. tubing before any connections are made.

2. Open Petri dish containing assembled chamber and firmly connect maleend of i.v. extension tubing to outlet port hub. Cover the Petri dish tokeep the inlet port sterile.

3. Fill a 1 ml tuberculin syringe with 0.8 to 1.0 cc. of the desiredcell suspension.

4. Attach syringe to inlet port and slowly charge the chamber, beingcareful to eliminate all air bubbles by tilting the chamber so that theoutlet port is up.

5. Leave the syringe in place and position the chamber so that the cellssettle onto the desired coverglass'(upper coverglass for standardmicroscope, lower coverglass for inverted microscope). Incubate thechamber at 37 C. in this position for an appropriate period of time.

6. When ready to flush the chamber at the end of the settling period,fill a 20 ml. glass syringe with media, expelling all air bubbles.

7. Attach i.v. extension tubing to the syringe and fill tubing withmedia, again expelling all air bubbles.

8. Remove the tuberculin syringe from the inlet port and attach themedia-filled i.v. extension tubing.

9. Clear the system of air bubbles so that the only bubbles visible arein the distal end of the outlet tubing just before it enters the outletbottle.

10. Flush the chamber with an appropriate amount of media to remove allnon-adhering cells from the chamber (about 4 cc).

11. Place the syringe on a perfusion pump capable of sustaining a mediaflow rate of about 1 cc per hour.

The chamber is then placed on the mechanical stage plate of themicroscope for observation. This stage plate is preferably constructedof carbon steel, preferably plated, with a suitable material whichallows the use of magnetic clips to hold the chamber firmly in place.Example of suitable plating material for the stageplate would be cadmiumor chromium. Subsequent ad- 6 ditions to the chamber (e.g., infectiousagents, radiochemicals, etc.) are made through the remaining input portof the Teflon 3-way valve.

A syringe infusion pump is used to continuously perfuse the chamber at aconstant rate with fresh media. The perfusion characteristics of thechamber are directly related to the flow rate of the media. A flow rateof approx. 1 ml/h results in a clean sweeping actionof the perfusatethrough the chamber. Lower flow rates result in diffusion of theperfusate in the chamber; higher flow rates result in vortical mixing.

Chamber temperature can be maintained with an air curtain incubator orsimilar device. The relatively large mass of the steel holder andmechanical stage plate act as a thermal buffer, thus restrictingtemperature fluctuations to approx. 0.2 C.

The lower aperture of holder 11 should be large enough to allow theappropriate optical element of the microscope unrestricted access to thelower coverglass. The appropriate optical element may consist of acondenser in the case of an upright microscope or an objective in thecase of an inverted microscope. In one embodiment, the aperture is 19.0mm. in diameter. The upper opening of the holder 11 is 30.5 mm. diameterwith a total thickness of 8.9 mm. The coverglasses are 25 mm. indiameter the the spacer has a 19.0 mm. aperture. The Teflon layers areabout pm thick.

It is important to understand that the particular materials anddimensions set forth in this specification are only suggestedembodiments. Any non-toxic, biologically inert materials may be used andthose of ordinary skill in the art will readily be able to designdimensions which will retain the inventive concept of the presentinvention but will adapt it for other uses. It

is accordingly to be understood that changes may be made withoutdeparting from the invention.

What is claimed is:

1. A cell culture chamber for use with light microscopy techniques,comprising:

a holder having an aperture and a peripheral groove extending outwardlyfrom said aperture therein;

a first transparent cover means held by said holder and covering saidaperture;

a second transparent cover means within said holder and positioned abovesaid first transparent cover means;

rigid spacer means within said holder and between said first and secondtransparent cover means, for maintaining said first and secondtransparent cover means at a predetermined distance from one another;

a rigid pressure plate within said holder and contacting said secondtransparent cover means;

compression means within said holder and contacting said pressure platefor urging said pressure plate against said second transparent covermeans, said spacer means, said first transparent cover means, and saidholder;

uniform pressure applying means for applying uniform pressure to saidcompressing means, said uniform pressure applying means comprising asnap element fitting into said peripheral groove of said holder; and

at least two rigid tubes passing through said rigid spacing means forpassing fluid into and out of the chamber defined by said first andsecond transparent cover means and said spacer means.

2. A cell culture chamber in accordance with claim 1, wherein:

said first and second transparent cover means are made of glass havingno substantial strain or other optical defects. 3. A cell culturechamber in accordance with claim 2, wherein:

said holder, said spacer means, said pressure plate and said tubes areall made of stainless steel machined dry, and wherein said tubes andspacer are integral. 4. A cell culture chamber in accordance with claim3, wherein:

all metal surfaces which contact glass are coated with a thin layer ofprotective plastic. 5. A cell culture chamber in accordance with claim1, wherein:

said holder is generally round and said aperture therein is round, saidholder having a recess therein above, said aperture with a diametergreater than that of said aperture, and said groove is located on thewall of said recess; said first cover means which covers said aperturerests on the bottom of said recess; said spacer means, said pressureplate and said compression means are ring-shaped and the aperturesthereof are at least substantially as large as the aperture of saidholder; and said snap element comprises a snap ring. 6. A cell culturechamber in accordance with claim 1, wherein:

said holder has an alignment radius machined on its periphery foralignment with an alignment pin on a microscope stage, and said holderhas a number inscribed on both faces to allow rapid identification ofsaid holder. 7. A cell culture chamber in accordance with claim 1,further including:

hub means connected to each of said tubes for rapid and easy connectionwith tubes or syringes for feeding or carrying away material to or fromthe chamber. 8. A cell culture chamber in accordance with claim 7,further including:

hub holding means connected to said holder and attachable to said hubmeans for rigidly holding said hub means in place during operation.

1. A cell culture chamber for use with light microscopy techniques,comprising: a holder having an aperture and a peripheral grooveextending outwardly from said aperture therein; a first transparentcover means held by said holder and covering said aperture; a secondtransparent cover means within said holder and positioned above saidfirst transparent cover means; rigid spacer means within said holder andbetween said first and second transparent cover means, for maintainingsaid first and second transparent cover means at a predetermineddistance from one another; a rigid pressure plate within said holder andcontacting said second transparent cover means; compression means withinsaid holder and contacting said pressure plate for urging said pressureplate against said second transparent cover means, said spacer means,said first transparent cover means, and said holder; uniform pressureapplying means for applying uniform pressure to said compressing means,said uniform pressure applying means comprising a snap element fittinginto said peripheral groove of said holder; and at least two rigid tubespassing through said rigid spacing means for passing fluid into and outof the chamber defineD by said first and second transparent cover meansand said spacer means.
 2. A cell culture chamber in accordance withclaim 1, wherein: said first and second transparent cover means are madeof glass having no substantial strain or other optical defects.
 3. Acell culture chamber in accordance with claim 2, wherein: said holder,said spacer means, said pressure plate and said tubes are all made ofstainless steel machined dry, and wherein said tubes and spacer areintegral.
 4. A cell culture chamber in accordance with claim 3, wherein:all metal surfaces which contact glass are coated with a thin layer ofprotective plastic.
 5. A cell culture chamber in accordance with claim1, wherein: said holder is generally round and said aperture therein isround, said holder having a recess therein above said aperture with adiameter greater than that of said aperture, and said groove is locatedon the wall of said recess; said first cover means which covers saidaperture rests on the bottom of said recess; said spacer means, saidpressure plate and said compression means are ring-shaped and theapertures thereof are at least substantially as large as the aperture ofsaid holder; and said snap element comprises a snap ring.
 6. A cellculture chamber in accordance with claim 1, wherein: said holder has analignment radius machined on its periphery for alignment with analignment pin on a microscope stage, and said holder has a numberinscribed on both faces to allow rapid identification of said holder. 7.A cell culture chamber in accordance with claim 1, further including:hub means connected to each of said tubes for rapid and easy connectionwith tubes or syringes for feeding or carrying away material to or fromthe chamber.
 8. A cell culture chamber in accordance with claim 7,further including: hub holding means connected to said holder andattachable to said hub means for rigidly holding said hub means in placeduring operation.